CELLULAR PATHOPHYSIOLOGY OF ACUTE RENAL FAILURE

缺氧对新鲜分离的近端小管损伤的研究 在目前的资助期内，我们认识到一个新的、巨大的规模， 甘氨酸和一种有限的代谢非依赖性的细胞保护作用 结构上相关的小分子中性氨基酸的数量。我们也 扩大了我们先前的观察，即中度酸中毒对 近端小管细胞。我们假设甘氨酸在人体内的可用性 结合细胞内pH的变化起着主要作用， 可能是决定急性肾小管细胞改变的关键因素 细胞内pH在细胞内起着主要作用，可能是决定性的作用。 缺氧状态下急性肾小管细胞活性的测定 复氧损伤，包括导致肾小管细胞损伤 到体内的缺血性急性肾功能衰竭。甘氨酸是最重要的 这两个因素之间的关系，并可能对其他各种因素产生影响 伤害设置和细胞类型也是如此。在下一个资助期内， 使用新鲜分离的近端小管和培养的小管 上皮细胞，我们计划进行研究以进一步了解损伤 甘氨酸具有保护作用的条件，以确定哪些因素 最终限制甘氨酸的细胞保护，并阐明其特异性 甘氨酸的细胞靶点S。为此，我们有五个具体目标： 1)用改进的方法测定细胞内游离钙(Caf) 可能与损伤状态最相关的低微摩尔范围，我们 计划更准确地确定CAF的入职要求 发生的磷脂水解和根尖膜重塑 尽管甘氨酸具有细胞保护作用，并评估这些过程是否 进一步受到低pH条件的调节。此外，我们还将聘用 共聚焦显微镜和生化技术研究的贡献 细胞骨架改变对这种膜重塑的影响及其作用 在甘氨酸细胞保护方面。2)我们将扩展我们对氧气的分析 剥夺及相关形式的ATP耗竭所致的损伤 分析致死性膜损伤发生的机制 尽管甘氨酸含量高，pH值低。3)我们将评估 一种对甘氨酸高度敏感的复氧损伤的机制 孤立的近端小管。4)我们发现甘氨酸可以 保护新鲜分离和培养的动物免受氧化损伤 小管细胞，但保护比其他形式的更有选择性 伤害和可能不表达的铁介导的损伤对新鲜 近端小管。我们将确定甘氨酸的适用性 对氧化剂致肾小管损伤的细胞保护作用。5)我们已经展示了 甘氨酸在MDCK细胞损伤过程中的细胞保护作用 并且已经能够克隆出差异敏感的品系。我们会 描述克隆敏感度变异的基础，尝试 隔离具有更多所需属性的其他行，并使用 这些线，我们将扩大我们的结构活性研究的保护 化合物并开始工作，以描绘特定的细胞骨架 蛋白质是甘氨酸的主要靶标。我们的总体目标仍然是 了解急性肾功能衰竭的细胞病理生理学 预期这将导致改进预防和预防方法 治疗。甘氨酸细胞保护是一种非常强大的工具 为此目的，对肾脏和其他 器官

In studies of hypoxic injury to freshly isolated proximal tubules during the current grant period, we recognized a novel, large magnitude, metabolism-independent, cytoprotective action of glycine and a limited number of structurally-related small neutral amino acids. We also extended our prior observation that moderate acidosis is protective for proximal tubule cells. We hypothesize that availability of glycine in combination with alterations of intracellular pH plays a major role, possibly the deciding one, in determining acute tubule cell alterations of intracellular pH plays a major role, possibly the deciding one, in determining acute tubule cell viability during oxygen deprivation- reoxygenation injury, including the tubule cell damage which contributes to ischemic acute renal failure in vivo. Glycine is the most important of the two factors and can potentially impact on a variety of other injury settings and cell types as well. During the next funding period, using both freshly isolated proximal tubules and cultured tubule epithelial cells, we plan studies to further understand the injury conditions under which glycine is protective, to determine what factors ultimately limit glycine cytoprotection, and to elucidate the specific cellular target(s) of glycine. To these ends we have five Specific Aims: 1) Using improved methods for assessing cytosolic free calcium (Caf) in the low micromolar range that may be most relevant to injury states, we plan to more precisely determine the Caf requirements for induction of the phospholipid hydrolysis and apical membrane remodeling that occurs despite glycine cytoprotection and to assess whether these processes are further modulated by low pH conditions. Additionally, we will employ confocal microscopy and biochemical techniques to study the contribution of cytoskeletal alterations to this membrane remodeling and their role in glycine cytoprotection. 2) We will extend our analysis of oxygen deprivation and related forms of ATP depletion-induced injury in isolated tubules to analyze the mechanisms for lethal membrane damage that occurs despite high glycine and low pH conditions. 3) We will assess the mechanisms of a highly glycine-sensitive form of reoxygenation injury to the isolated proximal tubules. 4) We have found that glycine can be protective against oxidant injury to both freshly isolated and cultured tubule cells, but protection is more selective than for other forms of injury and may not be expressed against Fe-mediated damage to the fresh proximal tubule. We will determine the applicability of glycine cytoprotection to oxidant-induced tubule injury. 5) We have demonstrated strong expression of glycine cytoprotection during injury to MDCK cells and have been able to clone differentially sensitive lines. We will characterize the basis for clonal variation in sensitivity, attempt to isolate additional lines with even more desirable properties, and, using these lines, we will expand our structure activity studies of protective compounds and initiate work to delineate whether specific cytoskeletal proteins are the primary targets for glycine. Our overall aim remains to understand the cellular pathophysiology of acute renal failure in the expectation that this will lead to improved methods of prevention and treatment. Glycine cytoprotection is an extraordinarily powerful tool for this purpose with implications for injury to the kidney and other organs